The broad objective of this research program is to understand how basal forebrain (BF) cholinergic (BFC) and non-cholinergic neurons are organized to modulate specific cortical regions. Despite its involvement in cortical activation, attention, and memory, the functional details of the BF are not well understood due to the anatomical complexity of the region. Patients with Alzheimer's disease and related dementias have a significant decrease of acetylcholine in the cortex and show pathological changes in cholinergic neurons in the BF. Thus, a complete understanding of its functional organization is warranted. The central hypothesis of this application is that cholinergic neurons constitute local ensembles in the BF ('cell clusters') that via local collaterals and/or common inputs with their projections to cortical areas provide the neural basis of a distributed functional network to selectively modulate cognitive processes. We will test this hypothesis in 4 interrelated Specific Aims using traditional and monosynaptic viral tracing, computational analysis of large-scale networks, in vitro patch-clamp recording of BF neurons, and high-resolution monitoring of cortical network activity in freely-moving rats with optogenetic stimulation of defined BF cholinergic neurons. In Aim 1 we will build up a relatively complete database with 200 ?m resolution of mapped BF cholinergic and non-cholinergic neurons using conventional retrograde tracing techniques. Cholinergic clusters will be defined in the resulting 'database' and in the cluster volume significant association of projection cell populations will be determined. In Aim 2 we will validate of the functional significance of the specific organization of the BFC system in wake-behaving rats. With newly developed multi-array silicon probes implanted into two specific cortical areas and light-assisted perturbation of various cholinergic cell groups in ChAT-Cre rats, in which cholinergic cells were transfected to express channelrhodopsin (ChR2), we will determine the emerging cholinergic ensembles in the BF and their effect on the functional connectivity of various large-scale cortical networks during various brain states. In Aim 3 we will define the input to cholinergic neurons in various subdivisions of the BF using commercially available Cre-dependent AAV helper viruses (AAV-EF1a-FLEX- TVAmCherry and AAV-CA-FLEX-RG) and a replication deficient rabies vector (RV: EnvA G-deleted Rabies- eGFP). In Aim 4 we will determine, using in vitro patch clamp recording and retrograde tracing in ChAT-cre X ChAT-eGFP crossbred mice, how does the system of early (EF) and late firing (LF) cholinergic neurons and local cholinergic axon arborizations fit into the global organization of the BFC system. The in vivo large-scale, high-density recording design that is built on a realistic forebran model will lead to substantially improved animal models for addressing function in behavioral studies. Concomitantly, it will facilitate the understanding of the aberrant processing in basalo-cortical networks and may help the development of new treatment strategies to ameliorate the cognitive symptoms in Alzheimer's and related disorders.